Volume 20, Issue 15, Pages 1378-1382 (August 2010) Rabex-5 Ubiquitin Ligase Activity Restricts Ras Signaling to Establish Pathway Homeostasis in Drosophila  Hua Yan, Maryam Jahanshahi, Elizabeth A. Horvath, Hsiu-Yu Liu, Cathie M. Pfleger  Current Biology  Volume 20, Issue 15, Pages 1378-1382 (August 2010) DOI: 10.1016/j.cub.2010.06.058 Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 1 Loss of Rabex-5 Causes Increased Size, Ectopic Wing Veins, and Eye/Antennal Phenotypes (A and B) FRT80B control, left. Homozygous Rabex-5 deletion (ex42/ex42) resulted in giant larvae (A, right) and pupae (B, right), often containing melanotic masses (arrows, A). (C) Constitutive Rabex-5 RNA interference (RNAi) (Act>Rabex-5IR, right) increased adult size compared to Actgal4/+ controls (left, males shown). (D) Table of Act>Rabex-5IR phenotypes. Length, weight, and wing area are normalized to 1.000 for controls and are shown as a ratio to controls ± standard deviation; numbers of samples are in parentheses. (E) Overlay of Act>Rabex-5IR (red) and control Actgal4/+ (blue) wings. (F) Control Actgal4/+ wing. (G) Act>Rabex-5IR wings contain extra posterior crossveins (PCV, bottom arrow) and extra longitudinal veins (LV, top arrow). (H) Control c765gal4/+ wing. (I and J) Rabex-5 RNAi in the wing at 30°C using c765gal4 caused extra veins (arrows, I) and was enhanced by deletion allele Rabex-5ex42 (J). (K) Table of wing phenotypes. Wings with ectopic veins over total wings are shown in parentheses. (L) Control ey>dcr2 eye. (M–O) Strong Rabex-5 RNAi in the early eye (ey>dcr2, Rabex-5IR) produced overgrown eyes (M), small eyes (N), extra antennae (O, overhead view of N) and other abnormalities (Figure S1). (P) Table of eye results. For tables in (D), (K), and (P), ∗p < 0.05 compared to control; †p < 0.05 for suppression by Ras alleles. Images and quantitation shown in (D)–(O) are from females; quantitation in (P) is of males. (Q) Western blot of larvae homozygous for control FRT80B or Rabex-5ex42. dpERK (top) and total ERK (bottom) staining showed 3.9-fold ± 0.23 (standard deviation) increased ERK activation in mutant larvae compared to controls. Additional controls are shown in Figure S1. Current Biology 2010 20, 1378-1382DOI: (10.1016/j.cub.2010.06.058) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 2 Loss of Rabex-5 Enhances Activated Egfr and Oncogenic Ras (A–C) Activated Egfr, EgfrA887T (left, A), or RasV12S35 (left, B) expressed in differentiating eye cells caused small, rough eyes. (A and B) Rabex-5 RNAi (right) enhanced GMR>EgfrA887T (A) and GMR>RasV12S35 (B), as evidenced by the appearance of black tissue (arrows; enlarged in insets) and lethality (Figure S2). (C) Table quantifying effects of Rabex-5 loss on EgfrA887T, RasV12S35, and Rafgof. Female eyes are shown and quantified. In Rafgof eyes, Rabex-5 reduction did not statistically significantly change the frequency of black tissue compared to control (p > 0.05). (D–G) ELAV (green) stains presumptive photoreceptors; Distal-less (red) stains presumptive antennae in larval discs. (D) eygal4/+ control. (E) ey>Rabex-5IR control. (F) Larval disc expressing RasV12S35 in the early eye. (G) RasV12S35 discs with reduced Rabex-5 showed increased overgrowth, extra antennae (arrowheads), and ectopic eyes (arrow). Scale bar represents 200 μm. (H) Graph quantifying phenotypes in (D) and (E). For table in (C) and graph in (H), ∗p < 0.05 compared to control. Additional controls and quantitation are shown in Figure S2. Current Biology 2010 20, 1378-1382DOI: (10.1016/j.cub.2010.06.058) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 3 Rabex-5 Regulates Wing Vein Specification via Its Ubiquitin Ligase Domain (A) Control engal4/+ wing. (B and C) Expressing Rabex-5WT (B) or Rabex-5DPYT (C) using engal4 resulted in crossvein loss (arrows). (D) Expressing Rabex-5FY did not alter wing vein pattern. Female wings are shown. (E) Table of results from (A)–(D). ∗p < 0.05 for transgenes compared to control. (F–H) Wing clones expressing Rabex-5DPYT (green) show loss of dpERK staining (red in G and H, arrows in G). (H) Merge of (F) and (G). Scale bar represents 50 μm. Additional controls are shown in Figure S3. Current Biology 2010 20, 1378-1382DOI: (10.1016/j.cub.2010.06.058) Copyright © 2010 Elsevier Ltd Terms and Conditions

Figure 4 Rabex-5 Ubiquitin Ligase Domain Suppresses Oncogenic Ras (A–C) Coexpressing Rabex-5WT (A, RasV12S35+Rabex-5WT, right) or Rabex-5DPYT (B, RasV12S35+Rabex-5DPYT, right) in differentiating eye cells suppressed RasV12S35 (left in A–C). (C) Coexpressing Rabex-5FY (RasV12S35+Rabex-5FY, right) did not suppress RasV12S35. (D) Summary table. Eye size was normalized to 1.000 for GMRgal4/+ controls and is shown as a ratio to controls ± standard deviation. ∗p < 0.05 for Rabex-5 and/or RasV12S35 transgene expression compared to GMRgal4/+ control; †p < 0.05 for suppression of GMR>RasV12S35. (E) Nickel pull-down of lysates from cells expressing tagged Ras, Ub, and Rabex-5. Rabex-5 expression increased Ras ubiquitination (right lane). Anti-HA (top) and anti-FLAG (bottom) staining are shown. Diubiquitinated Ras is indicated by arrows; unconjugated Ras is indicated by an arrowhead. Additional controls are shown in Figure S4. Current Biology 2010 20, 1378-1382DOI: (10.1016/j.cub.2010.06.058) Copyright © 2010 Elsevier Ltd Terms and Conditions